Project Summary Effective cellular quality control is crucial for cellular homeostasis and cardiac health. Defects in the pathways that regulate the removal of damaged proteins and organelles contribute to heart failure. Autophagy is well known for its role in removing protein aggregates and organelles; however, it has recently been found that the small GTPase Rab5 and early endosomes also participate in Parkin-mediated mitochondrial clearance. Beclin1 is a scaffolding protein which can form distinct PI3K protein complexes to regulate different processes, such as autophagy. Although Beclin1 is well known as a regulator of autophagy, it has recently been implicated as a regulator of the endosomal degradation pathway. However, the mechanism underlying Beclin1?s regulation of the endosomal pathway remains unclear. In this proposal, we will explore the hypothesis that Beclin1 and UVRAG regulate the endosomal degradation pathway by forming a unique protein complex in the cell, which can be recruited by the active form of Rab5 under conditions of stress to enhance endosomal activity. This hypothesis will be tested with two aims. In Aim 1, we will demonstrate that Beclin1 and UVRAG form a unique protein complex to regulate Rab5 and endosomal activity in vitro. Using mouse embryonic fibroblasts that lack Beclin1, we will confirm that Beclin1 forms a stable complex with UVRAG that functions as a reservoir for active Rab5. Additionally, we will demonstrate that GTP-bound Rab5 positive endosomes recruit Beclin1/UVRAG to increase endosomal activity. In Aim 2, we will investigate the functional role of the Beclin1/UVRAG complex in vivo. Using an inducible cardiac-specific Beclin1 knockout mouse model, we will characterize how the loss of Beclin1 affects cardiac structure and function at baseline. Additionally, we will confirm the unique physiological role of a Beclin1/UVRAG complex by comparing these mice to Atg7 knockout mice, which are solely autophagy deficient. These studies will provide novel insights into how Beclin1 is able to regulate the endosomal degradation pathway and its importance in the heart. We will also gain an important new understanding of the physiological roles both the endosomal and autophagy degradation pathways may play in contributing to the prevention of heart failure.